SPHINGOSINE 1 PHOSPHATE IN EARLY BLOOD VESSEL FORMATION

Medical University Of South Carolina, Charleston SC

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Abstract

DESCRIPTION (provided by applicant): Blood vessels form through two distinct processes, vasculogenesis and angiogenesis. Vasculogenesis is the de novo formation of blood vessels from mesodermal precursor cells whereas blood vessels form from pre-existing vessels in the process of angiogenesis, Vasculogenesis was once thought to be operative exclusively in embryos, but now the process is now known to contribute to blood vessel formation in adults (e.g., in corpus luteal and endometrial neovascularization and in pathological neovascularization associated with tumors, cutaneous wounds and collateral coronary vessel formation). Indeed, adult neovascularization must now be considered a composite of vasculogenic and angiogenic processes. This realization has placed new emphasis on understanding the distinctions between the two processes and identifying the factors that influence each of these processes separately and together. While a considerable number of factors have been identified that modulate the process of angiogenesis, few have been found that influence vasculogenesis or that influence both processes. Sphingosine-1-phosphate (S1P) is a phosphorylated sphingolipid whose signaling via Edg receptors has been implicated in angiogenesis. However, the role of S1P in vasculogenesis has not been investigated. Our data show that sphingosine kinase (SPK), the enzyme responsible for S1P biosynthesis, and S1P receptors, Edg-1 and Egd-3, are expressed in embryonic tissues undergoing vasculogenesis. Furthermore, using a novel in vitro model of vasculogenesis, the murine allantois explant culture system, we show that S 1P is required to sustain neovascularization (at stages prior to the appearance of smooth muscle cells [SMCs]). These findings are consistent with the fact that Edg-1-null mice die early in development (12.5-14.5 days post coitum, dpc) due to vascular insufficiencies. Interestingly, the Edg-1-deficient phenotype includes inadequate SMC investment of blood vessels. Taken together these findings raise the possibility that both endothelial cells (ECs) and SMC are dependent on S1P. Given the fact that these cell types both arise from mesodermal progenitors, it is also possible that progenitor cells represent the critical target of S1P. The precise basis for the dependence remains uncertain since S1P is documented to elicit a range of cellular responses including inhibition of apoptosis and opposing effects on cell-cell adhesion/motility in ECs versus SMCs. The central hypothesis for experiments outlined in this application is that S1P is a critical signaling molecule for both vasculogenesis and angiogenesis. It is speculated that S1P influences definable growth and behavior characteristics of mesodermal progenitors of ECs and mural cells (pericytes and vascular SMCs) as well as mature ECs and SMCs engaged in embryonic blood vessel development. Although these studies emphasize determining the role of S1P in embryonic blood vessel formation, the findings are expected to have direct bearing on adult neovascular events as well. To test the above hypotheses there are three specific aims: 1) To define morphogenic steps in embryonic blood vessel formation at which S1P signaling plays a role. It is expected that discernable phases in the processes of neovascularization will be altered in embryos derived from mice deficient in S1P signaling owing to disruption in Edg-1/Edg-3-genes. 2) To determine the mechanism by which S1P affects embryonic blood vessel formation. Modulation of S1P signaling in a novel model of embryonic neovascularization is expected to lead to measurable alterations in the growth/survival, differentiation and/or motility of each of the major cellular constituents of the neovascular process (e.g., mesodermal precursors of endothelial and vascular SMCs, ECs and vascular SMCs). Results from these studies will lead to an understanding of which cells are influenced by S 1P signaling and the underlying mechanisms. 3) To determine the pathways regulated by and downstream targets of S1P during embryonic blood vessel formation. Specific biochemical pathways downstream of S1P will be defined by comparing gene expression profiles of Edg-1/Edg-3-deficient and normal embryos.